Project Summary Eukaryotic cells have developed sophisticated defenses aimed at limiting viral replication and thereby preventing infection from escalating to other cells. Viperin (Virus Inhibitory Protein; Endoplasmic Reticulum associated, INterferon inducible) is one of the many interferon-stimulated genes whose expression is up- regulated in the antiviral response. Viperin has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV, cytomegalovirus and hepatitis C. Interestingly, viperin is one of only 8 radical SAM enzymes identified in humans. Although viperin has been known for some time, it was only recently that viperin was discovered to catalyze the formation of the antiviral nucleotide 3'-deoxy-3',4'-didehydro- CTP (ddhCTP) by dehydration of CTP. ddhCTP acts as a chain-terminating inhibitor to disrupt replication of some RNA viral genomes, but not all ? and so ddhCTP synthesis cannot fully explain the wide spectrum of viperin's antiviral properties. It has also been shown that viperin interacts with wide variety of cellular and viral proteins and that these interactions are an equally important component of viperin's antiviral properties. The goal of this proposal is to develop a unified understanding of how viperin's seemingly disparate functions contribute to the antiviral response by investigating, at the molecular level, the interactions of viperin with the diverse classes of enzymes that it recognizes. In studies employing transiently expressed enzymes in HEK293T cells we have recently demonstrated that viperin interacts with various enzymes to either activate or repress their catalytic activity. In some cases, viperin also decreases the cellular half-lives of these enzymes. We now aim to understand how interactions between viperin and its partner enzymes regulate the activity of both viperin and the enzyme bound to it. The proposed studies will focus on: a) viperin's interaction with the mitochondrial trifunctional protein which plays and important role in the infective cycle of cytomegalovirus and a newly-recognized role in regulating thermogenesis; b) viperin's interaction with the E3 ubiquitin ligase TRAF6 and the interleukin receptor-associated kinase IRAK1; interactions between these enzymes are implicated in innate immune signaling through the TLR7/9 pathways and upregulation of viperin activity. To extend our understanding of how viperin regulates enzyme activity, we will investigate the interactions of viperin with cholesterol biosynthetic enzymes that we have provisionally identified by proteomic analysis. Viperin-induced down-regulation of cholesterol biosynthesis has been shown to inhibit enveloped viruses such as influenza A from budding from the cell membrane, which requires cholesterol-rich lipid rafts. We will reconstitute the complexes of viperin with the various in enzymes in vitro using purified enzymes and undertake detailed enzyme kinetic analyses, combined with biophysical measurements of protein and ligand binding, to study to study the mechanism(s) of activation or inhibition. Concurrently, we will use crystallography and/or cryo-EM, as appropriate, to determine structures for the complexes of viperin with these enzymes.